Analysis of Flavonoids in Dalbergia Odorifera by Ultra-Performance Liquid Chromatography with Tandem Mass Spectrometry

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Article Analysis of Flavonoids in Dalbergia odorifera by Ultra-Performance Liquid Chromatography with Tandem Mass Spectrometry

Xiangsheng Zhao 1, Shihui Zhang 1, Dan Liu 2, Meihua Yang 1,3 and Jianhe Wei 1,3,*

1 Hainan Provincial Key Laboratory of Resources Conservation and Development of Southern Medicine, Hainan Branch of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Haikou 570311, China; [email protected] (X.Z.); [email protected] (S.Z.); [email protected] (M.Y.) 2 School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang 621010, China; [email protected] 3 Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China * Correspondence: [email protected]; Tel.: +86-10-57833358

Received: 14 December 2019; Accepted: 15 January 2020; Published: 17 January 2020

Abstract: Dalbergia odorifera, a traditional Chinese medicine, has been used to treat cardio- and cerebrovascular diseases in China for thousands of years. Flavonoids are major active compounds in D. odorifera. In this paper, a rapid and sensitive ultra-high performance liquid chromatography-triple quadrupole mass spectrometry method was developed and validated for simultaneous determination of 17 flavonoids in D. odorifera. Quantification was performed by multiple reaction monitoring using electrospray ionization in negative ion mode. Under the optimum conditions, calibration curves for the 17 analytes displayed good linearity (r2 > 0.9980). The intra- and inter-day precisions (relative standard deviations) were lower than 5.0%. The limit of quantitation ranged from 0.256 to 18.840 ng/mL. The mean recovery range at three spiked concentrations was 94.18–101.97%. The validated approach was successfully applied to 18 samples of D. odorifera. Large variation was observed for the contents of the 17 analytes. Sativanone and 30-O-methylviolanone were the dominant compounds. The fragmentation behaviors of six flavonoids were investigated using UPLC with quadrupole time-of-flight tandem mass spectrometry. In negative ion electrospray ionization mass spectrometry, all the flavonoids yielded prominent [M H] ions. Fragments for losses of CH , CO, and CO were − − 3 2 observed in the mass spectra. Formononetin, liquiritigenin, isoliquiritigenin, sativanone, and alpinetin underwent retro-Diels–Alder fragmentations. The proposed method will be helpful for quality control of D. odorifera.

Keywords: Dalbergia odorifera; ﬂavonoid; assay; fragmentation behavior; UPLC-MS/MS

1. Introduction Dalbergia odorifera T. Chen (Leguminosae) is a semi-deciduous perennial tree that is indigenous to Hainan Province, South China. It has been introduced to and cultivated in Guangdong, Guangxi, Fujian, and Yunnan provinces, China [1]. Heartwood of D. odorifera is an important traditional Chinese medicine called “Jiangxiang” that is widely used to resolve stasis, stanch bleeding, regulate qi, and relieve pain [2]. In Korea, this heartwood is also used for treatment of blood stagnation, ischemia, swelling, necrosis, and rheumatic pain [3]. In addition, D. odorifera is commonly used as a component of commercial drug mixtures for cardiovascular treatment, including Guan-Xin-Er-Hao decoction [4], Qi-Shen-Yi-Qi decoction [5], Xinning tablets [2], and Tongxinluo capsules [6]. Also known

Molecules 2020, 25, 389; doi:10.3390/molecules25020389 www.mdpi.com/journal/molecules Molecules 2020, 25, 389 2 of 16 as fragrant rosewood (Huanghuali in Chinese), D. odorifera is a valuable wood product for manufacture of furniture, artifacts, and musical instruments [7]. Due to its high medicinal and commercial value, many researchers have studied D. odorifera. The strong market demands combined with the slow growth of D. odorifera have resulted in production of counterfeit items. To ensure the safety and efficiency of D. odorifera in clinical practice, quantitation of its functional components is critical. Phytochemical investigations have demonstrated that flavonoids and volatile oils are the main medicinal components of D. odorifera [8]. Flavonoids are secondary polyphenolic metabolites occurring commonly in many medicinal plants. Due to their extensive pharmacological activities, flavonoids are considered as the active principle components in many herbs. Recent investigations have shown that flavonoids in D. odorifera possess various biological activities, such as anti-inflammatory [9,10], antioxidant [11], antitumor [12], antibacterial [13], and vasorelaxant activities [14]. Meanwhile, 30-O-methylviolanone, sativanone, butein, liquiritigenin, butin, formononetin, etc. are the main compounds in D. odorifera [15–17]. Therefore, flavonoids could be considered as marker compounds to assess the quality of D. odorifera. However, no quantitative markers, except the content of its essential oils, have been selected for quality control of D. odorifera in the Chinese Pharmacopeia, which severely limits its clinical application and in-depth study. Among the analytical methods used for determination of flavonoids, the most widely used are based on high-performance liquid chromatography (HPLC) coupled with an ultraviolet (UV) or diode array detector [15–17]. The D. odorifera matrix is highly complex and the compounds of interest might be present in only minute quantities or accompanied by many other compounds with similar structures. In most cases, techniques like HPLC-UV will not be the optimum choice and can have long run times. A rapid, validated, and sensitive multi-component analytical method for quantification is required. Ultra-high performance liquid chromatography (UHPLC) combined with triple quadrupole mass spectrometry (QqQ-MS) is considered one of the most efficient techniques for quantitative analysis, and can provide specific, sensitive, and selective quantitative results in multiple reaction monitoring (MRM) mode [18]. Although numerous UHPLC-tandem mass spectrometry (MS/MS) methods have been applied to the determination of bioactivities components in traditional Chinese medicines [19,20], few studies have applied this method to quantitative analysis of flavonoids in D. odorifera [15–17,21]. Additionally, UHPLC-quadrupole time-of-flight (Q/TOF)-MS/MS has become increasingly important in compound identification because of its high selectivity, specificity, and accuracy [22]. In the present study, a rapid and sensitive UHPLC-QqQ-MS method was established using MRM mode for the simultaneous quantitative analysis of 17 flavonoids (daidzein, dalbergin, 30-hydroxydaidein, liquiritigenin, isoliquiritigenin, alpinetin, butein, naringenin, butin, prunetin, eriodictyol, tectorigenin, pinocembrin, formononetin, genistein, sativanone, and 30-O-methylviolanone, Figure1) in D. odorifera grown in different areas of China. The fragmentation behaviors of six different types of flavonoids were explored using UHPLC-Q/TOF-MS/MS in negative ion mode. This study is an example of comprehensive quality control and expands the knowledge of quantitative and qualitative analysis of multiple flavonoids in D. odorifera.

2. Results and Discussion

2.1. Method Development To develop a sensitive and accurate quantitative method, the analytes and internal standard (IS) were separately infused into the instrument to optimize the mass conditions. MS spectra were investigated in both positive and negative modes. All analytes showed maximum sensitivity in negative ion mode. For optimization of the MRM conditions, the cone voltage and collision voltage were optimized to acquire the richest relative abundance of precursor and daughter ions. Two transitions were monitored for identiﬁcation of each component, and the transition with the higher intensity was selected for quantiﬁcation. The retention time and MS parameters for each analyte are presented in Table1. Molecules 2020, 25, 389 3 of 16 Molecules 2019, 24, x FOR PEER REVIEW 3 of 18

FigureFigure 1. Chemical 1. Chemical structures structures of of the the seventeen seventeen target target compounds. compounds. 1, 3′ 1,-Hydroxydaidein; 30-Hydroxydaidein; 2, Butein; 2, Butein;3, 3, Daidzein;Daidzein; 4, 4, Liquiritigenin; Liquiritigenin; 5, 5, Eriodictyol; Eriodictyol; 6, Butin; 6, Butin; 7, Naringenin; 7, Naringenin; 8, Genistein; 8, Genistein; 9, Tectorigenin; 9, Tectorigenin; 10, Alpinetin; 11, Isoliquiritigenin; 12, Formononetin; 13, Dalbergin; 14, 3′-O-methylviolanone; 15, 10, Alpinetin; 11, Isoliquiritigenin; 12, Formononetin; 13, Dalbergin; 14, 30-O-methylviolanone; 15, Sativanone;Sativanone; 16, Pinocembrin; 16, Pinocembrin; 17, 17, Prunetin. Prunetin. Analytes Analytes numbers in in the the test test is the is the same same as in as this in figure. this ﬁgure.

To2. Results optimize and theDiscussion chromatographic behavior, the UHPLC conditions were explored. First, a Waters Acquity BEH C18 column (100 mm 2.1 mm, 1.7 mm) and Waters Acquity HSS T3 2.1. Method Development × (100 mm 2.1 mm, 1.8 µm) were examined. The Waters Acquity HSS T3 was chosen as it gave better × separationTo and develop sharper a sensitive peaks. and Next, accurate acetonitrile–water, quantitative method, methanol–water, the analytes and and internal various standard additives (IS) (i.e., were separately infused into the instrument to optimize the mass conditions. MS spectra were formic acid and acetic acid) were tested as potential mobile phases. Compared with the methanol–water investigated in both positive and negative modes. All analytes showed maximum sensitivity in system,negative the acetonitrile–water ion mode. For optimization system gave of the better MRM peak conditions, shapes the and cone resolutions. voltage and For collision the modifiers, voltage we foundwere that aceticoptimized acid to markedly acquire inhibitedthe richest the relative responses abundance of the compounds.of precursor and In addition, daughter ionization ions. Two of the flavonoidstransitions was inhibitedwere monitored if the concentrationfor identification of of formic each component, acid was too and high. the transition Therefore, with the the concentration higher of formicintensity acid was selected set at 0.05%. for quantification. The effects ofThe the retent columnion time temperature, and MS parameters flow rate, for and each elution analyte procedure are were alsopresented investigated. in Table 1. Finally, acetonitrile containing 0.05% formic acid with a flow rate of 0.25 mL/min To optimize the chromatographic behavior, the UHPLC conditions were explored. First, a and the 40 ◦C column temperature were selected to achieve satisfactory separation in a short time (FigureWaters2). Acquity BEH C18 column (100 mm × 2.1mm, 1.7 mm) and Waters Acquity HSS T3 (100 mm × 2.1 mm, 1.8 μm) were examined. The Waters Acquity HSS T3 was chosen as it gave better separation 2.2. Optimizationand sharper peaks. of the ExtractionNext, acetonitrile–water, Conditions methanol–water, and various additives (i.e., formic acid and acetic acid) were tested as potential mobile phases. Compared with the methanol–water system, Samplethe acetonitrile–water preparation system methods gave are better of key peak importance shapes and resolutions. in the analysis For the of modifiers, samples withwe found complex matrices,that acetic and especiallyacid markedly in inhibited the simultaneous the responses analysis of the compounds. of multiple In compounds. addition, ionization To develop of the an efficientflavonoids and appropriate was inhibited extraction if the method concentration for the 17of targetformic components acid was too for high. UHPLC-MS Therefore,/MS analysis,the refluxconcentration extraction and of formic ultrasonic acid was extraction set at 0.05%. were The compared effects of used the column sample temperature, S9 (0.2 g). The flow two rate, extraction and methodselution gave procedure similar were results also but investigated. the ultrasonic Finally, extraction acetonitrile was containing more convenient 0.05% formic (Figure acid with S1). a Thus, flow rate of 0.25 mL/min and the 40 °C column temperature were selected to achieve satisfactory ultrasonic extraction was chosen for subsequent experiments. To optimize the extraction, the extraction separation in a short time (Figure 2). solvent (50%, 60%, 70%, or 80% methanol, v/v), extraction volume (15, 20, 25, or 30 mL), and extraction time (30, 45, or 60 min) were investigated. When the methanol concentration was increased from 50–70%, the extraction efficiencies for the analytes increased (Figure3A). However, when the methanol concentration was increased beyond 70%, the extraction efficiencies showed no large increases. Therefore, we chose 70% methanol as the extraction solvent. There were no obvious differences in the contents of analytes between extraction volumes of 25 and 30 mL (Figure3B), and the contents of some compounds some compounds (e.g., eriodictyol, naringenin, 3 -O-methylviolanone, sativanone, 0 and pinocembrin) were higher than 20 mL. The best extraction time for all components was 45 min (Figure3C). Hence, the optimum conditions for extraction of D. odorifera were 0.2 g of dried sample, 25 mL of 70% methanol, and ultrasonic extraction for 45 min. Molecules 2020, 25, 389 4 of 16 Molecules 2019, 24, x FOR PEER REVIEW 4 of 18

Figure 2. Ultra-high performance liquid chromatographychromatography (UHPLC)-MS(UHPLC)-MS/MS/MS multiple reaction mode chromatograms of mixed standards (upper)(upper) andand samplesample (lower,(lower, S9).S9).

2.2. Optimization of the Extraction Conditions

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Table 1. MS/MS parameters for 17 target compounds.

Collision Collision No. Compounds Ion Mode RT (min) Precursor Ion Cone Voltage (V) Product ion 1 Q Product Ion 2 I Energy (eV) Energy (eV) - 1 30-Hydroxydaidein ESI 4.97 269 52 213 28 241 24 2 Butein ESI- 5.12 271 35 135 20 253 17 3 Daidzein ESI- 5.95 253 51 208 31 224 26 4 Liquiritigenin ESI- 6.23 255 40 135 15 119 22 5 Eriodictyol ESI- 6.24 287 38 151 17 135 24 6 Butin ESI- 7.59 271 39 135 31 91 37 7 Naringenin ESI- 7.94 271 40 151 20 119 24 8 Genistein ESI- 8.04 269. 48 133 32 181 28 9 Tectorigenin ESI- 8.30 2989 40 284 19 240 22 10 Alpinetin ESI- 8.65 269 44 165 20 227 21 11 Isoliquiritigenin ESI- 9.62 255 35 135 15 119 23 12 Formononetin ESI- 10.08 267 45 252 22 223 25 13 Dalbergin ESI- 10.09 267 38 180 27 252 18 - 14 30-O-methylviolanone ESI 10.62 329 46 135 38 299 36 15 Sativanone ESI- 11.10 299 46 135 37 269 32 16 Pinocembrin ESI- 12.47 255 42 107 25 171 25 17 Prunetin ESI- 12.97 283 45 268 21 239 26 18 Rutin (IS) ESI- 4.05 609 62 300 52 271 50 Q: transitions for quantiﬁcation; I: transitions for identiﬁcation. Molecules 2020, 25, 389 6 of 16 Molecules 2019, 24, x FOR PEER REVIEW 7 of 18

FigureFigure 3. 3.EffectsEﬀects of of(A ()A solvent) solvent concentration, concentration, (B ()B solvent) solvent volume, volume, and and (C ()C extraction) extraction time time on on the the extractionextraction efficiency eﬃciency of oftarg targetet analytes analytes in inS9 S9 sample. sample.

2.3. Method Validation

Molecules 2020, 25, 389 7 of 16

2.3. Method Validation The developed UHPLC-MS/MS method for quantitation of 17 flavonoids was validated to determine the specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), intra- and inter-day precisions, stability, and accuracy according to International Conference on Harmonization (ICH) guidelines for validation of analytical procedures [23].

2.3.1. Speciﬁcity The representative MRM chromatograms of the mixed standard solution and real sample solution are shown in Figure2. All of the target compounds could be distinguished using their retention times and precursor-to-product ion transitions. This indicates that the assay for D. odorifera is highly speciﬁc and selective.

2.3.2. Linear range, LOD, and LOQ Linearity was evaluated using the coeﬃcients of correlation (r2), which are listed along with the calibration curve equations, linear ranges, LOD, and LOQ in Table2. Within the investigated concentration ranges, all compounds showed good linearity with r2 ranging from 0.9986 to 0.9999. The LOD and LOQ for each analyte were calculated using signal-to-noise ratios of three and ten, respectively. The LOD range was 0.085–6.080 ng/mL and the LOQ range was 0.256–18.840 ng/mL for the 17 target compounds, which showed the method had high sensitivity.

2.3.3. Precision, Repeatability, and Stability The intra- and inter-day variability were measured to assess the precision of the developed method using sample 9. The intra-day precision was evaluated by analyzing six replicates prepared from sample 9, and the inter-day precision was examined over three consecutive days with samples per day. The repeatability was determined by injection of six samples prepared following the same procedure (Section 2.4). The stability of the sample solution over 24 h at room temperature was also evaluated. For the precision, repeatability, and stability tests, the percent relative standard deviations were within 5.0% (Table2).

2.3.4. Accuracy To further evaluate the accuracy of the proposed method, a recovery test was carried out by spiking three levels (80%, 100%, and 120% of the known amount) of mixture standard solution and corresponding IS standards to known amount samples. Next, the spiked samples were extracted and analyzed using the proposed method, and then, triplicate experiments were performed at each level. The recoveries were calculated using the following equation: Recovery (%) = (total amount detected − amount in original sample)/amount spiked 100%. The recovery for each compound was in the range × of 94.18–101.97% and the relative standard deviation was less than 6.0% (Table S1 in Supplementary Materials). The results implied that the developed UHPLC-MS/MS was precise, accurate, sensitive, and reliable enough for simultaneous quantitative analysis of the 17 target compounds in D. odorifera. Molecules 2020, 25, 389 8 of 16

Table 2. Curves, test range, limit of detection (LOD), limit of quantiﬁcation (LOQ), precision, and repeatability for the seventeen analytes.

Precision (RSD, %) Repeatability No. Compounds Calibration Curves r2 Linear Range (ng/mL) LOQ (ng/mL) LOD (ng/mL) Intra-Day Inter-Day (RSD, %, n = 6) 1 3 -Hydroxydaidein Y = 0.934X 0.0436 0.9991 5.40–1350 5.400 1.600 2.43 3.23 3.45 0 − 2 Butein Y = 0.1675X 0.198 0.9993 1.41–2820 1.410 0.470 2.49 4.85 4.53 − 3 Daidzein Y = 0.9697X 0.061 0.9989 3.02–1510 3.020 1.000 1.74 3.01 2.98 − 4 Liquiritigenin Y = 0.1813X + 0.0075 0.9999 1.61–3220 1.610 0.500 1.25 2.26 3.18 5 Eriodictyol Y = 0.1802X 0.0166 0.9997 1.36–1360 1.360 0.453 2.38 2.45 2.06 − 6 Butin Y = 0.1163X 0.0568 0.9986 1.51–3020 1.510 0.458 1.85 3.54 2.77 − 7 Naringenin Y = 0.2291X 0.0722 0.9989 2.72–1360 2.720 0.906 2.07 4.61 4.06 − 8 Genistein Y = 0.8139X 0.2152 0.9988 3.82–1910 3.820 1.528 0.76 3.33 2.54 − 9 Tectorigenin Y = 0.203X 0.2161 0.9987 2.44–1220 2.440 0.813 1.96 2.40 1.95 − 10 Alpinetin Y = 0.5127X 0.0544 0.9996 5.36–1340 5.360 1.790 2.85 4.94 4.76 − 11 Isoliquiritigenin Y = 0.1308X + 0.0284 0.9996 1.416–1770 1.416 0.480 0.45 3.02 3.67 12 Formononetin Y = 0.0516X 0.0608 0.9993 0.516–1290 0.516 0.172 1.78 1.90 3.32 − 13 Dalbergin Y = 0.2867X 0.0665 0.9991 0.256–1280 0.256 0.085 3.51 4.85 4.61 − 14 30-O-methylviolanone Y = 0.6244X + 0.0119 0.9989 9.90–2970 9.900 3.300 2.08 4.49 2.87 15 Sativanone Y = 0.675X 0.1047 0.9991 18.84–5652 18.840 6.080 1.24 1.26 4.73 − 16 Pinocembrin Y = 0.3485X 0.0569 0.9992 2.66–1330 2.660 0.870 1.04 1.94 3.82 − 17 Prunetin Y = 0.0489X 0.0657 0.9989 1.12–2240 1.120 0.374 1.91 3.34 3.61 − Molecules 2020, 25, 389 9 of 16

2.4. Method Application The validated method was applied to determine the 17 selected flavonoids in 18 samples of D. odorifera. Representative MRM chromatograms are shown in Figure2 and the quantitative results are shown in Table3. The contents of the 17 analytes varied in di fferent batches of D. odorifera. Sativanone and 30-O-methylviolanone were the dominant compounds in D. odorifera. The content of sativanone in all batches ranged from 5.8806 to 24.1200 mg/g (4.10-fold variation), and that of 30-O-methylviolanone ranged from 0.6973 to 7.583 mg/g (10.87-fold variation). The content of daidzein in most samples was lower than the LOQ. For 30-hydroxydaidein, genistein, and alpinetin, the contents were also relatively low (<0.2 mg/g). The trends observed in our results were similar to those in previous studies [15–17]. For example, Liu et al. [15] analyzed 10 major flavonoids in D. odorifera by HPLC-UV and found that sativanone (1.45–20.90 mg/g) was dominant. The average contents of other flavonoids (e.g., liquiritigenin, formononetin, and dalbergin) were higher than our results. In another study, seven flavonoids were analyzed in D. odorifera by Li et al. [17] and the obtained concentration ranges for the detected analytes (liquiritigenin, formononetin, isoliquiritigenin, and naringenin) were similar to those in the present study. Variation in the levels of flavonoids among the samples could be caused by differences in geographical conditions, the tree ages, plant origins, and storage conditions. The results suggest that UHPLC-MS/MS is a very powerful technique for quantitative analysis of multiple components of D. odorifera because it is rapid and sensitive.

2.5. Fragmentation Pathways Analysis To date, 99 flavonoids have been isolated from D. odorifera [21]. These compounds have the same basic skeleton with different substituents. A total of 17 flavonoids, including six isoflavones (30-hydroxydaidein, daidzein, genistein, tectorigenin, formononetin, and prunetin), five flavanones (liquiritigenin, eriodictyol, butin, naringenin, and pinocembrin), two chalcones (butein and isoliquiritigenin), two isoflavanones (sativanone and 30-O-methylviolanone), one flavone (alpinetin), and one neoflavone (dalbergin) were quantified in the present study. Negative ion electrospray ionization (ESI) mode was found to be more sensitive than positive ion mode for detecting flavonoids. To further identify the compounds in D. odorifera, fragmentation pathways of six representative flavonoids (formononetin, pinocembrin, isoliquiritigenin, sativanone, alpinetin, and dalbergin) of D. odorifera were examined by UPLC-Q/TOF-MS/MS in negative ionization mode. Formononetin is a methoxylated isoflavone. The suggested fragmentation pathway of formononetin is shown in Figure4a. The main and typical fragmentation ions of this compound result from successive or simultaneous losses of CH3, CHO, CO, and CO2, which are attributed to the 40-OCH3 isoflavone type [24]. The base peak ion of formononetin at m/z 252.0491 [M H-CH ] is formed by − 3 − loss of a CH3 group. This result is consistent with a previous study that showed that loss of CH3 was characteristic of fragmentation in methoxylated flavonoids [25]. As the collision energy increased, abundant characteristic fragment ions were observed at m/z 223.0437 [M H-CH -CHO] , m/z 224.0480 − 3 − [M H-CH -CO] , m/z 208.0563 [M H-CH -CO ] , m/z 195.0491 [M H-CH -CHO-CO] , m/z − 3 − − 3 2 − − 3 − 180.0627 [M H-CH -CO -CO] , and m/z 167.0543 [M H-CH -CHO-2CO] . It is worth mentioning − 3 2 − − 3 − that neutral loss of CO2 is common for isoflavones in MS/MS and the fragment ion at m/z 223.0437 differed from m/z 267.0666 by 44 Da, which is typically assigned as neutral loss of CO2. However, the TOF/MS revealed that the formula of m/z 223.0437 was C14H7O3, and this was formed by loss of C2H4O rather than CO2. Therefore, the typical loss of CO2 did not occur in this case. Instead, this fragment was produced via losses of CH3 and CHO at the 40-position [26]. Fragment ions at m/z 132.0259 [1,3B H] and 135.0125 [1,3A H] were produced by retro-Diels–Alder (RDA) fragmentation − − − − in the C-ring of formononetin. Molecules 2020, 25, 389 10 of 16

Table 3. Contents of 17 analytes in 18 batches of samples (mg/g).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 S1 0.1004 0.0769 0.0546 0.7228

FigureFigure 4.4.MS MS/MS/MS spectra spectra and theand proposed the proposed fragmentation fragme pathwayntation ofpathway formononetin of formononetin (a), liquiritigenin (a), (liquiritigeninb), and isoliquiritigenin (b), and isoliquiritigenin (c). (c).

Liquiritigenin gave a precursor ion [M H] at m/z 255.0649 (Figure4b). In the MS /MS spectrum, The MS/MS spectra and fragmentation− −pathway of sativanone are shown in Figure 5a. characteristicGenerally, losses ions of were CH3 observed and CO2 were at m/ zprominent.134.9958, 119.0398 Loss of CH and3 from 93.0264, the whichB-ring wereof sativanone consistent yielded with the typical [1,3A H] and [1,3B H] fragments. Isoliquiritigenin showed similar fragmentation fragments at m/z− 284.0719− [M − H-CH− −3] and m/z 269.0158 [M − H-CH3] produced from the precursor behaviorion at 299.0566 to liquiritigenin ([M − H]−). (Figure Loss of4c). CO2 from m/z 269.0158 yielded [M − H-2CH3-CO2]− (m/z 225.1346). A fragmentThe MS /ionMS at spectra m/z 134.9958 and fragmentation [1,3A − H]− was pathway generated of sativanone after RDA are cracking, shown inand Figure further5a. loss Generally, of CO2 lossesfrom m of/z CH 134.99583 and CO produced2 were prominent. [1,3A − H-CO Loss2]− at of m CH/z 91.0106.3 from the Additionally, B-ring of sativanone fragmentation yielded at fragmentsthe C-ring at m/z 284.07190,3 − [M H-CH ] and m/z 269.0158 [M H-CH ] produced from the precursor ion at produced a B ion− at m/z 179.05823 with low abundance.− Further3 loss of one H2O produced an ion at m/z 161.0083. This fragmentation pathway was consistent with the previous report of Zhao et al. [27].

Molecules 2020, 25, 389 12 of 16

299.0566 ([M H]−). Loss of CO2 from m/z 269.0158 yielded [M H-2CH3-CO2]− (m/z 225.1346). − 1,3 − A fragment ion at m/z 134.9958 [ A H]− was generated after RDA cracking, and further loss of CO2 1,3 − from m/z 134.9958 produced [ A H-CO2]− at m/z 91.0106. Additionally, fragmentation at the C-ring 0,3 − produced a B− ion at m/z 179.0582 with low abundance. Further loss of one H2O produced an ion at Moleculesm/z 161.0083. 2019, 24 This, x FOR fragmentation PEER REVIEW pathway was consistent with the previous report of Zhao et al.14 of [27 18].

Figure 5. MS/MS spectra and the proposed fragmentation pathway of sativanone (a), alpinetin (b), Figureand dalbergin 5. MS/MS (c). spectra and the proposed fragmentation pathway of sativanone (a), alpinetin (b), and dalbergin (c). The fragmentation behavior for alpinetin is shown in Figure5b. Alpinetin gave a [M H] ion − − at m/Thez 269.0820 fragmentation as the base behavior peak. for Two alpinetin radical anionsis shown at min/ zFigure254.0539 5b. [MAlpinetinH-CH gave] anda [M 225.1623 − H]− ion [M at − 3 − m/z 269.0820 as the base peak. Two radical anions at m/z 254.0539 [M − H-CH3]− and 225.1623 [M − H-CO2]− formed by loss of CH3 and CO2 from the precursor anion. Additionally, a peak at m/z 165.0222 [1,3A − H]− was observed after RDA fragmentation. Little research has been conducted on the fragmentation pathways of neoflavones [28]. The mass spectrum of dalbergin (Figure 5c) exhibited significant ions at m/z 267.0655, 252.0166, 224.1377, and 180.0407. The precursor ion lost one CH3 to give an ion at m/z 252.0166. Subsequent loss of CO from this ion generated a fragment ion m/z 224.1377. Further loss of CO2 from m/z 224.1377 yielded a fragment ion at m/z 180.0407.

Molecules 2020, 25, 389 13 of 16

H-CO ] formed by loss of CH and CO from the precursor anion. Additionally, a peak at m/z − 2 − 3 2 165.0222 [1,3A H] was observed after RDA fragmentation. − − Little research has been conducted on the fragmentation pathways of neoflavones [28]. The mass spectrum of dalbergin (Figure5c) exhibited significant ions at m/z 267.0655, 252.0166, 224.1377, and 180.0407. The precursor ion lost one CH3 to give an ion at m/z 252.0166. Subsequent loss of CO from this ion generated a fragment ion m/z 224.1377. Further loss of CO2 from m/z 224.1377 yielded a fragment ion at m/z 180.0407. In negative ion ESI-MS/MS, all target analytes yielded prominent [M H] ions. Some common − − features, such as loss of CH3, CO, and CO2, were observed in the MS/MS spectra, and were consistent with the literature. The [M H-CH ] ion was a characteristic fragment of methoxylated flavonoids − 3 − (formononetin, alpinetin, and dalbergin). In addition, [M H-2CH ] fragments were observed − 3 − for dimethoxylated flavonoids (sativanone). Therefore, loss of one or two CH3 could be adopted to identify single- or multi-methoxylated flavonoids in negative ion ESI-MS/MS. Loss of CO and CO from [M H] ions was attributed to the structure of the C-ring. The [M H] ions of 2 − − − − formononetin, liquiritigenin, isoliquiritigenin, sativanone, and alpinetin underwent RDA fragmentation. However, RDA fragmentation was not observed for dalbergin, which may be related to its specific structural characteristics.

3. Materials and Methods

3.1. Solvents and Chemicals HPLC-grade acetonitrile and formic acid were purchased from Thermo Fisher Scientific (Fair Lawn, NJ, USA). Analytical grade methanol was purchased from Beijing Chemical Works (Beijing, China). Deionized water was prepared using a Milli-Q system (Millipore, Milford, MA, USA). Reference standards of daidzein, dalbergin, 30-hydroxydaidein, liquiritigenin, isoliquiritigenin, alpinetin, butein, naringenin, butin, prunetin, eriodictyol, and tectorigenin were purchased from Chengdu Chroma-Biotechnology Co., Ltd. (Chengdu, China). Rutin for use as an internal standard (IS), pinocembrin, formononetin, and genistein were obtained from Sichuan Victory Biological Technology Co., Ltd. (Chengdu, China). The purities of all standards were above 98.0%. We isolated sativanone and 30-O-methylviolanone from the heartwood of D. odorifera T. Chen. The structures of these two compounds were unambiguously identified by NMR techniques, and their purities were determined to be above 96% by HPLC. Heartwood samples of Dalbergia odorifera T. Chen (n = 18) were collected from different areas in China. The samples were identified by Prof. Jianhe Wei (Institute of Medicinal Plant Development, Chinese Academy of Medical Science & Peking Union Medical College, Beijing, China), and voucher specimens (No. 469027-LD-020) were deposited in the Resource Center for Chinese Materia Media (Hainan Branch Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, China).

3.2. UHPLC-QqQ-MS/MS Analyses were performed on a UHPLC system (Acquity H-Class, Waters Corp., Milford, MA, USA) with a binary solvent manager, a column manager, and a sample manager. The sample was separated on a Waters Acquity HSS T3 column (100 mm 2.1 mm, 1.8 µm; Waters Corp.) and × the column temperature was set at 40 ◦C. The mobile phase consisted of acetonitrile (A) and water containing 0.05% formic acid (B) with a flow rate of 0.25 mL/min. The following gradient program was used: 5–30% A from 0–2 min, 30–33% A from 2–5 min, 33–53% A from 5–13 min, held at 53% A for 3 min, 53–95% A from 16–18 min, held at 95% A for 2 min, and 95–5% A from 20–22 min. For UHPLC-MS/MS analysis, a Waters QqQ-MS (Xevo TQ-D, Waters Corp.) was connected to the Waters UHPLC instrument via an electrospray ionization (ESI) source. Analytes were quantified by MRM in negative ionization mode with argon as the collision gas. All MS parameters were optimized in Molecules 2020, 25, 389 14 of 16 the combined mode. The following ESI ion source parameters were used: capillary voltage, 2.5 kV; source temperature, 150 ◦C; desolvation temperature, 400 ◦C; cone gas (nitrogen) flow rate, 50 L/h; and desolvation gas (nitrogen) flow rate, 600 L/h. The UHPLC-MS/MS parameters, including the precursor-to-product ion transitions, cone voltage, and collision energy, are listed in Table1.

3.3. UHPLC-Q/TOF-MS/MS Flavonoid fragmentation was performed on a quadrupole time-of-flight mass spectrometer (Xevo G2-XS, Waters Corp.) equipped with an ESI source and coupled to the UPLC system. The above UHPLC conditions were used for UHPLC-Q/TOF-MS/MS. Detection was implemented in the MSE centroid mode over a mass range of 500–1000 Da with a scan rate of 10 Da/s. The analyzer sensitivity mode was used. Leukine enkephalin was infused using LockSpray via a reference probe for in-run mass corrections. The ESI ion source parameters were as follows: capillary voltage, 2.5 kV; desolvation gas (nitrogen) flow rate, 600 L/h, desolvation temperature, 400 ◦C; cone gas flow rate, 50 L/h; and source temperature, 150 ◦C. The collision energy was ramped in a high energy function from 20 to 60 eV using argon as the collision gas. MassLynx software (Waters Corp.) was used for post-acquisition analysis.

3.4. Sample Preparation The materials were pulverized and dried to a constant mass before use. A 0.20 g sample was extracted with 25 mL of 70% methanol (v/v) in an ultrasonic water bath for 45 min and then filtered. An aliquot (1 mL) of the filtrate was transferred into a 15-mL screw cap plastic tube containing 9 mL of 70% aqueous methanol and shaken immediately for 1 min using a vortex mixer. Then, 0.5 mL of IS (1.0 µg/mL) was added to 0.5 mL of the solution and the mixture was vortexed for 30 s. The obtained solution was filtered through a 0.22-µm micropore membrane before use. A 5 µL sample was injected into the UHPLC instrument for analysis.

3.5. Standard Solution Preparation Standard stock solutions of 17 reference standards with a concentration range of 24.4 to 113.04 µg/mL were separately prepared by dissolution in methanol. An initial stock solution was prepared as a mixture of the above stock solutions. A series of working solutions of the analytes were obtained by diluting the mixed stock solution with methanol to the appropriate concentration. Then, 0.5 mL of IS (1.0 µg/mL) was added to 0.5 mL of the working solutions and the mixture was vortexed for 30 s. All of the solutions were stored at 4 ◦C and ﬁltered through a 0.22-µm nylon membrane before injection into the UHPLC system.

4. Conclusions In the present study, a sensitive and rapid UHPLC-ESI-MS/MS method was established for the simultaneous quantitative analysis of 17 flavonoids in the heartwood of D. odorifera and successfully applied to 18 samples. Satisfactory validation parameters were obtained, including specificity, linearity, precision, accuracy, and stability, and the extraction method was optimized. Taking the contents of the target compounds into consideration, the quantification results indicated that the quality of D. odorifera varied. The MS fragmentation pathways of flavonoids discussed here could facilitate rapid screening and structural characterization of these compounds by LC-MS/MS. Our results suggest that UHPLC-MS/MS could be a useful tool for quality assessment of D. odorifera using flavonoids as markers.

Supplementary Materials: The following are available online, Table S1: Recoveries of 17 target compounds. Author Contributions: Conceptualization, J.W. designed the experiments; X.Z. performed the experiments and wrote the paper; S.Z. and D.L. optimized the condition of samples pretreatment. M.Y. contributed in polishing the language and grammer. All authors have read and agreed to the published version of the manuscript. Molecules 2020, 25, 389 15 of 16

Funding: This research was funded by the Key Research Project of Hainan Province, China (Grant No. ZDYF2018123) and the CAMS Innovation Fund for Medical Sciences (CIFMS) (Grant No. 2016-I2M-2-003). Conﬂicts of Interest: The authors declare that they have no conﬂict of interest.

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Sample Availability: Samples of the compounds are not available from the authors.

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